Investigation of lipid metabolism by a new structured lipid with medium

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Investigation of lipid metabolism by a new structured lipid with medium and long-chain triacylglycerols from Cinnamomum camphora seed oil in healthy C57BL/6J mice Jiang-Ning Hu, Jin-rong Shen, Chao-Yue Xiong, Xue-Mei Zhu, and zeyuan deng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05659 • Publication Date (Web): 11 Feb 2018 Downloaded from http://pubs.acs.org on February 11, 2018

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Journal of Agricultural and Food Chemistry

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Investigation of lipid metabolism by a new structured lipid with

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medium and long-chain triacylglycerols from Cinnamomum

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camphora seed oil in healthy C57BL/6J mice

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Jiang-Ning Hu1,2*, Jin-Rong Shen2, Chao-Yue Xiong2, Xue-Mei Zhu1,2, Ze-Yuan

5

Deng2*

6 7

1

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116034, China

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2

10

School of Food Science and Technology, Dalian Polytechnic University, Dalian

State Key Laboratory of Food Science and Technology, Institute for Advanced Study,

Nanchang University, Nanchang, Jiangxi 330047, China

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Running title: MLCTs alter lipids metabolism in C57BL/6J mice

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* To Corresponding authors:

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Telephone No: +86 88304449-8226, E-mail address: [email protected] (J-N Hu);

16

Telephone/ Fax No: +86 791 88304402, E-mail address: [email protected] (Z-Y Deng)

17

1

ACS Paragon Plus Environment


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Abstract

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In the present study, a new structured lipid with medium and long-chain

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triacylglycerols (MLCTs) was synthesized from camellia oil (CO) and Cinnamomum

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camphora seed oil (CCSO) by enzymatic interesterification. Meanwhile, the

22

anti-obesity effects of structured lipid were investigated through observing the

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changes of enzymes related to lipid mobilization in healthy C57BL/6J mice. Results

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showed that after synthesis, the major triacylgeride (TAG) species of intesterifcatied

25

product changed to LaCC/CLaC (12.6 ± 0.46%), LaCO/LCL (21.7 ± 0.76%),

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CCO/LaCL (14.2±0.55%), COO/OCO (10.8±0.43%), and OOO (18.6±0.64%).

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Through second stage molecular distillation, the purity of interesterified product

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(MLCT) achieved to 95.6%. Later on, male C57BL/6J mice were applied to study

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whether the new structured lipid with MLCT has the efficacy of preventing the

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formation of obesity or not. After feeding with different diets for 6 weeks, MLCTs

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could reduce body weight and fat deposition in adipose tissue, lower plasma

32

triacyglycerols

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(4.03±0.08mmol/L), and

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30.5%, respectively, when compared to control 2 group. This was also accompanied

35

by increasing fecal lipids (113%) and the level of enzymes including cyclic adenosine

36

monophosphate (cAMP), protein kinase A (PKA), hormone-sensitive lipase (HSL),

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adipose triglyceride lipase (ATGL) related to lipid mobilization in MLCT group.

38

From the results, it can be concluded that MLCT reduced body fat deposition

39

probably by modulating enzymes related to lipid mobilization in C57BL/6J mice.

(TG)

(0.89±0.16

mmol/L),

plasma

total

cholesterol

(TC)

hepatic lipids (382±34.2 mg/mice)by 28.8%, 16.0%, and

2


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Keywords: Medium- and long-chain triacylglycerols, obesity, lipid mobilization,

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triacyglycerols, total cholesterol

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Introduction

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Obesity is one of common health problems all over the world which will increase the

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incidence of diabetes, hypertension and coronary heart diseases.1 Many factors may

46

lead to fat accumulation. For example, excess calories from high – fat diet is a major

47

contributory factor for its development.2 Therefore, strategies for treating obesity are

48

very necessary, such as exercise, medication and dietary fat restriction. However,

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Medications are always along with some adverse effects and exercise is often hard to

50

insist. Among these measures, dietary modification is supposed to a wise choice to

51

prevent and control obesity. 3

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Cinnamomum camphora (lauraceae) is an evergreen tree growing in Jiangxi province

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and the south area of the Yangtze River in China. It has been reported that

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Cinnamomum camphora (lauraceae) seeds oil (CCSO) is nontoxic and could be used

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to product MCT-enriched plastic fat.4 Our previous data showed that the oil from

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Cinnamomum camphora (lauraceae) seeds mainly contains medium-chain fatty acids

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(MCFA) (capric acid, C10:0, 53.27%; lauric acid, C12:0, 39.93%).5 Medium-chain

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fatty acids (MCFA), which contain 8-12 carbon atoms, are found in palm kernel and

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coconut oils. MCT that consist of three MCFA on the skeleton of glycerol has

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particular nutritional and physiologic properties compared with LCT in animal foods

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and edible vegetable oil.6 In contrast to LCT, MCT are hydrolyzed rapidly to MCFA,

3



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which do not need incorporate into chylomicron and pass through the lymphatic

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system and the portal blood, and then directly to the liver. Therefore, it is reasonable

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to make MCT candidates for the dietary treatment of obesity.7 Many researches have

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demonstrated the effects of MCT on reducing body weight, lowering lipoprotein

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secretion, and attenuating postprandial triglyceride response in animal and human

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studies.8-10 Nevertheless, MCT lack essential fatty acid and possess low smoking

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point which makes it difficult to be used to cooking oil.11 Hence, a new type of

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structured lipid called medium- and long-chain triacylglycerols (MLCT) was

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developed to overcome the drawbacks of MCT. MLCT containing LCFA and MCFA

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at the same glycerol molecule produced by transesterification technique can be used

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as cooking oil.12 In addition, several numerous studies showed that a diet rich in

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MLCTs, which had the semblable postprandial thermogenesis and fat accumulation as

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those of MCTs, could reduce cholesterol, restrain the accumulation of body fat and

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blood triacylglyceride upon consumption.13-15

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Camellia oil (CO) is a rich source of oleic acid (C18:1, 75–80%) derived from the

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seeds of C. oleifera, which is similar to olive oil. CO plays a prominent role in

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reducing triglyceride and cholesterol level in blood due to the high content of oleic

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acid.16 The purpose of this study was to produce MLCTs composed of CCSO and CO.

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Then, the hypothesis that a MLCT containing diet could decrease fat accumulation

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and prevent obesity compared with MCTs- enriched CCSO was investigated.

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Material and methods

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Chemicals

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Cinnamomum camphora seed oil (CCSO) was obtained from Cinnamomum

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camphora seeds by organic solvent extraction. Refined CO was purchased from local

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oil market (Nanchang, China). Lipozyme RM IM. A commercial immobilized lipase

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from Rhizomucor miehei was purchased from Novozymes A/S (Bagsvaerd, Denmark).

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#463 of standard fatty acid methyl esters (FAMEs) were purchased from Nu-Chek

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Prep Inc. (Elysian, MN, USA). Glycerol Tributyrate (purity >99%) regarded as TAG

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standard was purchased from Sigma Chemical Co., St. Louis, MO, USA. All solvents

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and reagents used in analyses were of chromatographic or analytical grade。

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Synthesis and purification

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The interesterified product was produced through synthesis of CCSO and CO via

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Lipozyme RM IM with the molar ratio of 1:1 at 60 °C for 3 h which modified on the

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basis of a previous study

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molecular distillation. The optimal conditions were chosen as follows: feeding rate, 1

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ml/min; operation pressure, 60 Pa; first stage evaporating temperature, 90 °C; second

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stage evaporating temperature, 170 °C; scraper speed 250 rpm/min.

[4]

. The purity of interesterified product was conducted by

100

High-performance liquid chromatography (HPLC)

101

The

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high-performance liquid chromatograph (HPLC) equipped with an evaporative

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light-scattering detector (Alltech 3300, Deerfield, IL, USA) operating at 40 °C and a

104

gas flow rate of 1.5 L min-1. The mobile phase consisted of (A) tert butyl methyl ether

105

and (B) hexane with a flow rate of 1.0 mL min-1. A linear solvent gradient was

purity

of

interesterified

product

was

analyzed

5


using

normal-phase


106

determined in a previous study.17 Twenty microliters of filtered sample were injected

107

into the column Hypersil BDS CPS (250 * 4.6 mm, Waters, Milford, MA, USA). Peak

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identification was determined by retention times using TAG standards.

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Analysis of fatty acid composition

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The fatty acid compositions were determined by gas chromatography with slight

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modifications based on a previous report.18 The samples were converted into fatty acid

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methyl esters (FAMEs) and then injected into a gas-liquid chromatograph (Model

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6890N; Agilent Technologies, Palo Alto, CA, USA) equipped with an auto injector

114

and a flame ionization detector (FID) using a fused silica capillary column (CP-Sil 88,

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100 m 9*0.25 mm * 0.2 ml i.d.). The injector and detector temperatures were

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maintained at 250 and 260 °C, respectively. The initial temperature of the oven was

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held at 45 °C for 3 min, and then programmed at a rate of 13 °C min-1 to 175 °C for

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27 min. The temperature was then further increased at the rate of 4 °C min-1 to 220 °C

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and held for 35 min. The carrier gas was nitrogen at a flow of 53 mL min-1. Fatty

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acids compositions were identified by comparison with relative retention times of

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FAME standards. All samples were analyzed in duplicate.

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Sn-2 positional analysis

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Fatty acid composition at sn-2 position was determined by pancreatic lipase

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analysis.19 Tris–HCl buffer (pH 7.6,10 ml), 0.05% bile salt (2.5 ml), 2.2% CaCl2 (1.0

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ml) and pancreatic lipase (10 mg) were added to each sample (10 mg) for hydrolysis.

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After mixing, the temperature of samples remained constant at 37 ℃ for 2 min and

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shaken three times. Hydrolytic products were extracted and separated by thin-layer

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chromatography (TLC) plate developed in hexane/diethyl ether/acetic acid (50:50:1,

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v/v/v). The band corresponding to 2-monoacylglycerol was scraped and then

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methylated. The fatty acid composition was analyzed by gas-liquid chromatography

131

(GLC).

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Experimental animals

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Three-four weeks old, male mice (C57BL/6J) weighing 21±2 g were divided into five

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groups. Mice were housed in a room at 25℃ with 12/12 light/dark cycles for six

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weeks. The composition of five different diets is given in Table 1. Body weight was

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measured every week and food consumption was recorded daily. Total feces from

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each mouse were collected at week 6, freeze-dried, and shredded before analysis. At

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the end of week 6, mice were anaesthetized under carbon dioxide environment. Blood

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was collected from cava vein, and serum was separated and stored at- 80 ℃. Organs

140

were collected, washed with saline and frozen at a -80°C, until assays analyzed. This

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experimental was approved by the Law of Animal Experiment, University of

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Nanchang.

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Determination of plasma lipids

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Plasma TC, TG, LDL-C, and HDL-C were analyzed at week six using commercial

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enzymatic kits from Infinity (Waltham, MA, USA) and Stanbio Laboratories (Boerne,

146

TX, USA).

147

Determination of hepatic and fecal lipids

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Hepatic and fecal lipids were analyzed with minor modification on the basis of

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previously described.20 Total lipids were extracted from 400 mg liver sample or 600

7



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mg feces with chloroform–methanol (2:1, v/v), and then were dried through nitrogen

151

gas. Total lipids were separated into total TG, and phospholipids (PL) using bonded

152

phase columns. The eluent containing TG, and PL were dried under a gentle stream of

153

nitrogen gas and methylated with 14% BF3- methanol solution. The fatty acid methyl

154

esters (FAME) were determined by a gas-liquid chromatograph. The hepatic TG and

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TC levels were determined using commercial Triglyceride GPO-PAP and Cholesterol

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CHOD-PAP kits (Jiancheng Bioengineering Institute, China).

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Adipose tissue HE staining

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HE staining was analyzed as previously described.21 Adipose tissues were fixed in 10%

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formalin and then performed to the histological study by dehydration (increasing

160

alcohol concentrations, from low concentration to high concentration of alcohol),

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imbedding by paraffin after mounting in xylene. Next, the paraffin sections were cut

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into 5-mm thick for HE staining. The sections were observed under an Olympus

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CX31 microscope and then taken a picture by an Olympus U-CMAD3 camera under a

164

magnification of 100 times.

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ELISA validation

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Hepatic and adipose tissues were employed in the ELISA validation. Six proteins

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involved in lipid metabolism were quantified by using commercially available kits

168

following the manufacturers' instructions: apolipoprotein B (Apo B, Abcam,

169

Cambridge, UK), apolipoprotein A (Apo A, Abcam, Cambridge, UK), adenosine

170

3',5'-cyclic monophosphate (cAMP, Abcam, Cambridge, UK), adipose triglyceride

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lipase (ATGL,Abcam, Cambridge, UK), hormone-sensitive lipase (HSL,Abcam,

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Cambridge, UK) and protein kinase A (PKA, Abcam, Cambridge, UK). Duplicate

173

analyses were performed.

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Statistical analysis

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The statistical analysis system software was used to perform statistical evaluation.

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Quantitative data were presented as mean ± standard error of the mean. One-way

177

analysis of variance (ANOA) followed by Fisher’s LSD test was performed to

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determine the significance of difference at p < 0.05 among the five groups.

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Results

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Fatty acid profile, TAG species and the purity of interesterified product

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The fatty acid compositions of CO, CCSO, interesterified product (MLCT) are shown

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in Table 2. The results revealed that CCSO mainly contained saturated fatty acids

184

(SFA, 96.3%). Medium-chain fatty acids (MCFA) including capric acid (53.0±0.29%)

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and lauric acid (41.3±0.47%) were the most abundant fatty acids in CCSO. However,

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CO contained appreciable amounts of oleic acid (C18:1, 80.3±1.87%) and linoleic

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acid (C18:2, 7.68±0.25%) which belong to unsaturated fatty acids (USFA,

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89.0±0.51%). The content of oleic acid (C18:1) in interesterified product mainly

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distributed at sn-2 positions (41.5±0.26%) were much higher (52.1±0.31%) than that

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in CCSO (3.04±0.44%), while interesterified product contained more amount of

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medium-chain fatty acids (MCFA, 35.7±0.54%) compared with CO. After synthesis,

192

the major TAG species of intesterifcatied product changed to LaCC/CLaC (12.6%±

193

0.46%), LaCO/LCL (21.7% ± 0.76%), CCO/LaCL (14.2% ± 0.55%), COO/OCO

9



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(10.8%±0.43%), and OOO (18.6%±0.64%) (data shown in Table 3). Figure 1

195

showed that the purity of interesterified product achieved 95.6%, after second stage

196

molecular distillation.

197

Body length, body weight, food intake and organ weights

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As shown in Table 4, all group had similar initial body weights (21.4-21.6 g) at 0

199

week. As for food intakes (4.21-4.33 g), there were no significantly differences in the

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five groups across the whole experiment. After 6 weeks, High Fat (HF) group had a

201

final body weight of 28.0±1.26 g, which was significantly higher than other four

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groups (23.5-26.0 g). MLCT (24.10±0.99 g) which was similar to CCSO group

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(23.5±0.92 g) had lower final body weight than control 2 (26.0±0.29 g), and it had

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smaller epididymal and perirenal fat pads compared to HF and control 2 groups.

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Meanwhile, control 2 group (26.0±0.29 g) showed higher body weight than control 1

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(24.4±1.03 g) probably due to the consumption of cholesterol and custard powder.

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MLCT groups had smaller liver compared with HF groups.

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Plasma triacyglycerols (TG), total cholesterol (TC), HDL-C, LDL-C and

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HDL-C/LDL-C

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At the end of experimental period, group HF showed significantly higher

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triacyglycerols (TG) (2.22±0.04 vs. 1.25±0.06 mmol/L), total cholesterol (TC)

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(6.14±0.11 vs. 4.80±0.14 mmol/L), LDL-C (0.68±0.04 vs. 0.60±0.02 mmol/L) , as

213

well as lower HDL-C (2.59±0.03 vs. 3.01±0.05 mmol/L) than those in control 2 group.

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A significantly decrease in plasma TG (0.89±0.16 mmol/L ), total TC (4.03±0.08

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mmol/L), LDL-C (0.46±0.05 mmol/L) and a significantly increase in HDL-C

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(3.27±0.11 mmol/L ) were found in MLCT group compared with the control 2 group,

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resulting in an increase HDL-C/LDL-C from 5.02±0.56 to 7.11±1.81 in MLCT group

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(Table 5).

219

Fatty acids compositions of TG, PL, and total lipids in liver

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The total lipids and separated TG, PL were expressed as mg/mice. Adding lard into

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diet led to a significant increase in total lipids (Control 2: 549±47.1 vs. HF: 640±55.3

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mg/mice), TG (Control 2: 239±19.1 vs. HF: 326±20.1 mg/mice) and PL (Control 2:

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184±11.3 vs. HF: 239±17.1 mg/mice). However, dietary MLCT could decrease

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hepatic total lipids, TG and PL by 30.5%, 27.0%, 32.3% when compared with control

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2 group, respectively. (Table 6)

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Fecal lipids

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Table 7 showed the total fecal lipids, separated TG, and PL expressed as

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mg/mouse/day. The all total lipids, TG, and PL in HF group were decreased to

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2.00±0.01, 0.13±0.07, 0.27±0.03 when compared with control 2 group(4.00±0.03,

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0.17±0.08, 0.30±0.05), respectively. However, that adding MLCT into diet could lead

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to an increase in total fecal lipids (113%), separated TG (265%), and PL (30.0%) in

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the feces (MLCT vs. Control 2).

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Liver TG, TC, ApoA1, ApoB

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Table 8 revealed that HF group showed significantly higher hepatic TG content

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(31.5±2.6μmol/g liver ), TC content (16±2.36μmol/g liver) ) and apoB (40.2±0.37

236

ng/mg protein ) as well as lower content of apoA1 (12.9±0.95 ng/mg protein) than

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those in control 2 group. Administration of MLCT to diet in MLCT group for six

11



238

weeks resulted in a significant decrease in TG (17.7±3.1μmol/g liver ), TC (10.1±1.03

239

μmol/g liver ) and apoB (26.3±0.56 ng/mg protein) and a significant increase in

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apoA1 (18.2±1.36) when compared to control 2. Meanwhile, MLCT group showed

241

the highest level of apoA1/apoB (0.69). among the five groups.

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HE staining and enzymes related to lipid metabolism in adipose tissue

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Photomicographs of HE-stained adipose tissues are given in Figure 2. The size of

244

adipocytes of the HF group was significantly larger than the other four groups. ELISA

245

analyses showed that dietary MLCT increased the level of ATGL, HSL, cAMP and

246

PKA compared with HF group and control 2 group. HF group had the lowest level of

247

the four enzymes compared to the other four groups (Table 9).

248 249

Discussion

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As shown in Table 3, after interesterification, 41.5% of oleic acid was found at the

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sn-2 position of TAG. It has been known that LCFAs at sn-2 position of TAG are

252

conducive to digestion and absorption in the body. Moreover, MCFAs at sn-1,3

253

positions are beneficial for lowering body fat deposition and offering rapid energy.

254

Therefore, our MLCTs-enriched structured lipids could be developed as a cooking oil

255

contained nutritional value for food industries.4 To examine the effects of dietary

256

MLCT consumption on preventing obesity, we performed the trial on C57BL/6J mice

257

fed by meals containing MLCT compared to CCSO mainly composed of MCT and

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lard mainly composed of LCT for six weeks. In consequence, we found that mice fed

259

with MLCTs reduced body weight, decreased lipids of hepatic and adipose tissue and

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increased the levels of enzymes related to lipid metabolism than LCT group. These

261

results were in agreement with previous findings.1, 6, 14, 15 In recent years, many animal

262

and human researchers found that dietary MLCT led to a greater reduction of final

263

body weight than dietary LCT. For example, Takeuchi reported that the amount of

264

body fat in rats of MLCT group, which was fed by diet containing 4.8% (wt/wt)

265

MCFA for 6 weeks, was decreased compared with LCT group.22 Forty-eight male

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Wistar rats fed with MLCT containing 20% MCFA for 8 weeks lowered body weight

267

and fat accumulation in adipose tissue.11 A thorough study demonstrated that the

268

consumption of oil with medium- and long-chain TG in hypertriglyceridemic human

269

with BMI>22 kg/m2 had lower body weight, body fat percentage and subcutaneous

270

fat.6 These studies confirmed that shrinkage of fat depots mainly resulted in the

271

observed decreases in body weight.15, 23, 24

272

In this study, we found that mice fed with MLCT oil for 6 weeks resulted in a

273

significant reduction in blood TG in mice when compared with the fed of LCT oil.

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These results suggested that intake of MLCT oil may be a great treatment for

275

hypertriglyceridemic patients. The mechanism can be explained as (1) the peripheral

276

removal of circulating TG from blood, (2) hepatic production or secretion of TG into

277

blood. However, there have been few reports indicated that intake of MCT or MLCT

278

could decrease hepatic production of TG so far. What is more, there were many

279

reports indicative of the promoting effects of MCT or MLCT oil intake on energy

280

expenditure and fat oxidation in both liver and adipose tissue in animal and human

281

studies.25,26 In consequence, oxidation of fatty acids stimulated by intake of MCFAs

13



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282

might have contributed to our results. The results in many researches about the effects

283

of MCT on blood cholesterol metabolism are divergent. Zhang et al. reported that

284

blood cholesterol concentration after consumption of MLCT-supplemented diet for 8

285

weeks did not be affected when compared with those intake of LCT-supplemented

286

diet.15Hashim et al. showed in 8 healthy subjects that the consumption of an

287

MCT-supplemented diet for 2 weeks resulted in slight elevation in plasma total

288

cholesterol concentrations as compared with consumption of a corn oil-supplemented

289

diet.28 However, our studies indicated that MLCT consumption decreased blood

290

cholesterol concentration when compared with LCT consumption which was similar

291

to those of Michio et al.1 Previous studies reported that the reductase activity of a key

292

enzyme in cholesterol synthesis of hepatic called 3-hydroxy-3-methylglutaryl-CoA

293

(HMG-CoA) in rats was reduced after intake of an MCT containing diet, resulting in a

294

decrease of serum total cholesterol.29, 30 Hence, our results could be explained by

295

intake of MLCT in the diet led to a decrease in hepatic HMG-CoA reductase activity.

296

We also found that MLCT consumption increased HDL-C concentration and

297

decreased LDL-C level. These conclusions confirmed that MLCT may have beneficial

298

effects against cardiovascular diseases.

299

Many investigations have indicated that dietary MCT induced less fat deposition in [1, 31]

300

adipose tissue than LCT, which was consistent with our current studies

301

Especially the morphology of adipose tissues, the result showed that adipocytes from

302

LCT group were significantly larger than that in the MLCT group, indicating that the

303

effect of MLCT on adipose tissues metabolism was obvious. Adipose tissue is not

14


.

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only the main organ of triglyceride storage but also the target organ inducing obesity.

305

So, we select the adipose tissue as a target, observing the changes of enzymes related

306

to lipid metabolism. Hormone—sensitive lipase (HSL) was considered to be a sort of

307

major rate limiting enzymes relating to hydrolysis of triglyceride in adipose tissue.

308

But triglyceride was still hydrolyzed in the adipose tissue of the mouse defected HSL

309

gene which indicated that HSL is not the intracellular hydrolase for adipose lipid

310

mobilization.32 In 2004, some researchers almost simultaneously reported a newly

311

discovered enzyme - adipose triglyceride lipase (ATGL) with specific molecular

312

features and structures in adipose tissue. Further studies suggested that ATGL was

313

considered as the key enzyme of the first step in hydrolysis of triglycerides. Villena et

314

al. found that the gene expression of ATGL in obese individuals is regulated by some

315

adipokines.33 Because of the inhibition of ATGL in adipose tissue, the catabolism of

316

triglyceride was significantly reduced in-vitro assays which demonstrated that the

317

enzyme could play an important role in lipid metabolism.34 Previous study showed

318

that HSL had the ability to break down triglycerides by phosphorylation of protein

319

kinase A (PKA) depended on adenosine 3',5'-cyclic monophosphate (cAMP). Iemitsu

320

et al. confirmed that PKA was an essential factor for HSL while ATGL did not rely on

321

the activated PKA .35 Our studies indicated that the levels of cAMP, PKA, ATGL and

322

HSL in MLCT groups were higher than LCT group which further validated the

323

control of adipocyte differentiation by MLCT mainly through cAMP-PKA pathway.

324

After digestion in small intestine, MCT was hydrolyzed to MCFA which promoted

325

cAMP synthesis, increased the levels of PKA and promoted HSL phosphorylation.

15



326

MCFA might work on ATGL accompanied with HSL to promote TG hydrolysis.

327

Therefore, we speculated the mechanism of fat mobilization promoted by MLCT was

328

that MLCT increased steatosis by improving the level of enzyme related to adipose

329

lipid mobilization in adipose tissue.

330

Liver plays a vital role in fatty acid catabolism and synthesis. There have been few

331

reports focusing on apolipoprotein metabolism in subjects consuming oil containing

332

MLCT. Our studies showed that there were lower level of Apo B and higher level of

333

Apo A in MLCT group compared with LCT group. Apo A is a main carrier of high

334

density lipoprotein while Apo B is a main apolipoprotein of low density lipoprotein.

335

The levels with decrease of Apo A and increase of Apo B are considered as risk

336

factors for coronary heart diseases.36 The absence of apolipoproteins or their contents

337

are not fit to the increasing amount of serum lipids could lead to the lipid metabolic

338

disorders.37 In the present study, dietary MLCT decreased the accumulation of total

339

lipids, TG, and PL in the liver. Why MLCT consumption does reduce hepatic lipids

340

needs to be further determined. One possible explanation is that dietary MLCT could

341

down-regulate the expression of hepatic LPL which is responsible for the removal of

342

TG from plasma to the liver. Meanwhile, the intake of MLCT induced higher

343

concentration of fecal lipids as compared with the intake of LCT. These results

344

suggested that MLCT promoted fat absorption in the intestine. Therefore, the activity

345

of MLCT lowering body fat and preventing obesity was mainly due to its enhanced

346

lipid uptake by the liver and promoted its effect on the fat absorption.

347

In conclusion, a dietary intake of MLCT was found to have beneficial effects on

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348

helping prevent obesity, reducing body fat, and decreasing blood triglyceride,

349

cholesterol and LDL-C levels in C57BL/6J mice. The mechanism appears to be

350

explained by that MLCT might be useful for controlling lipid metabolism in hepatic

351

and fat absorption in the intestine. For future health promotion, MLCT is expected to

352

act as functional oil that can be widely used to prevent obesity as well as metabolic

353

disorders.

354 355

Acknowledgements

356

The authors acknowledge the National Key Research and Development Program of

357

China (2016YFD0401404) and National Natural Science Foundation of China (Grant

358

31571870, 31460427) for supporting this project.

17



359 360

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476 477

Table 1 Composition of five different diet. Percent of Control Control Nutrients 1 2 Protein (%) 18.0 12.6 Fat (%) 19.4 13.6 Carbohydrate (%) 48.5 33.9 Mineral mixture 0.78 0.54 (%) Vitamin mixture 0.52 0.36 (%) Fiber (%) 1.5 1.05 Water (%) 9.5 6.64 Others (%) 1.8 1.26 Cholesterol (%) 0 0.15 Custard powder 0 10 (%) Distilled water 0 20 (%) Lard (%) 0 0 Camphora seed oil 0 0 (%) Tea oil (%) 0 0 Medium and long 0 0 chain ester (%)

High Fat

CCSO

MLCT

12.6 13.6 33.9

12.6 13.6 33.9

12.6 13.6 33.9

0.54

0.54

0.54

0.36

0.36

0.36

1.05 6.64 1.26 0.15

1.05 6.64 1.26 0.15

1.05 6.64 1.26 0.15

10

10

10

0

0

0

20

0

0

0

20

0

0

0

0

0

0

20

478 479

24


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480 481 482

Table 2 Fatty acids composition (area%) of Cinnamomum camphora seed oil (CCSO), camellia oil (CO) and interesterified product (MLCT) CO CCSO MLCT Fatty acid Total sn-2 Total sn-2 Total sn-2 a a 8:0 ND ND 0.18 ±0.04 ND 0.06 ±0.00 0.22 ±0.02a 53.0±0.29e 64.4±0.61c 21.4 ±0.29e 26.1 ±0.38e 10:0 ND ND 32.1 41.3±0.47d 14.2 ±0.16d 16.8 ±0.29d b ±0.37 12:0 ND ND 0.80 1.24 ±0.03ab 0.45 ±0.07a ±0.10a 0.50 ±0.05a 14:0 ND ND 0.88 8.74±0.04b 1.68 ±0.02a 0.36 ±0.02a 6.19 ±0.12c 9.06 ±0.12c 16:0 ±0.21a 18:0 2.31±0.01a 0.57 ±0.10a 0.27 ±0.15a ND 0.16 ±0.03a 1.27 ±0.10a 1.15 85.0 ±0.91c 3.04 ±0.44bc 80.3±1.87c 52.1±0.31h 41.5±0.26f ±0.23a 18:1n-9 0.73 7.68 ±0.25b 12.2±0.22b 4.78 ±0.04b 4.07 ±0.41b 0.47±0.15a a 18:2n-6 ±0.04 20:1n-9 0.48 ±0.02a ND 0.08 ±0.01a ND 0.32 ±0.12a 0.09 ±0.02a 18:3n-3 0.47 ±0.07a 0.61±0.03a 0.07 ±0.02a ND 0.34 ±0.08a 0.39 ±0.01a b a f d Total SFA 11.1 ±0.45 2.25 ±0.17 96.3±0.73 98.1±0.82 42.5 ±0.26g 54.0 ±0.41h total 1.88 d d c USFA 89.0 ±0.51 97.8 ±0.58 3.66 ±0.06 ±0.16a 57.5 ±0.31i 46.0 ±0.52g total 96.4 MCFA ND ND 94.5 ±0.70f ±0.91d 35.7 ±0.54f 43.1±0.39f 483 484 485 486

Data are expressed as the mean ± SD; ND, not detected. Mean values in a column with different letters differ significantly, p < 0.05.

25



487 488

489 490 491 492 493

Page 26 of 36

Table 3 Triacylglycerol (TAG) composition (area%) of CCSO, CO, physical blend and interesterified product ECN

TAG

CCSO

CO

Interesterifie d Product

30 32 34 36 38 40 40 42 42 44 44 46 46 48 48 50

CCC LaCC/CLaC MCC/LaLaC CCO/LaCL LaCO/LCL LaLaO/LaOLa SLaC/PCL COO/OCO PPC/PCP/PCO/SCL LaOO/OLaO PLaO/PLaP/SLaL LOO/OLO PLO/PLP/LLS OOO POO/OPO SOO/OSO

2.27±0.12 84.5±1.24b 13.2±0.54b ND ND ND ND ND ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND ND ND ND 5.72±0.35b 1.27±0.10b 76.1±1.11b 15.7±0.56b 1.16±0.07

1.95±0.11 12.6±0.46a 5.19±0.34a 14.2±0.55 21.7±0.76 0.96±0.03 2.52±0.12 10.8±0.43 0.88±0.03 5.64±0.41 0.52±0.03 1.3±0.06a 0.23±0.02a 18.6±0.64a 2.79±0.10a 0.18±0.01

CCSO, camphora seed oil; CO, camellia oil; C, capric acid; La, lauric acid; M, myristic acid; P, palmitic acid; S, stearic acid; O, oleic acid; L, linoleic Acid; Ln, linolenic Acid; Equivalent carbon number (ECN) = Carbon Number - 2×Double Bonds; ND, not detected；Mean values in a column with different letters differ significantly, p < 0.05.

494

26


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Table 4 Changes of the body length, body weight, food intake and organ weights in C57BL/6J mice fed five different diets for six weeks Control 1 Control 2 High Fat CCSO MLCT Initial body 21.5±1.02 21.5±0.69 21.4±0.88 21.4±1.19 21.4±1.21 weight(g) Final body 24.4±1.03ab 26.0±0.29b 28.0±1.26c 23.5±0.92a 24.1±0.99a weight(g) Food intake(g/d) 4.21±1.83 4.25±1.31 4.33±1.01 4.28±1.65 4.28±1.35 Body length(cm) 8.43±0.27a 8.60±0.29ab 8.98±0.16b 8.26±0.18a 8.33±0.14a Organ weight(g) heart 0.12±0.01 0.14±0.02 0.14±0.01 0.13±0.03 0.13±0.02 Liver 0.82±0.09a 0.87±0.05ab 1.03±0.11b 0.84±0.09a 0.85±0.08a Spleen 0.05±0.00a 0.06±0.00ab 0.08±0.02c 0.07±0.01bc 0.06±0.00ab kidneys 0.30±0.03 0.31±0.02 0.31±0.01 0.31±0.03 0.30±0.02 Epididymal fat pad 0.35±0.09a 0.40±0.11ab 0.61±0.16b 0.34±0.09a 0.33±0.12a Perirenal fat pad 0.04±0.03a 0.04±0.04a 0.16±0.06b 0.04±0.02a 0.07±0.02a

495 496

497 498 499 500

Data are expressed as the mean ± SD. Mean values in a row with different letters differ significantly, p < 0.05.

27



501 502

Table 5 Change of triacyglycerols (TG), total cholesterol (TC), HDL-C, LDL-C and HDL-C/LDL-C in five experimental groups. Control 1 Control 2 High Fat CCSO MLCT a b c a TG(mmol/L) 0.99±0.12 1.25±0.06 2.22±0.04 0.92±0.09 0.89±0.16a TC(mmol/L) 4.04±0.22a 4.80±0.14b 6.14±0.11c 3.49±0.83a 4.03±0.08a HDL-C(mmol/L) 3.13±0.05bc 3.01±0.05ab 2.59±0.03a 3.29±0.21c 3.27±0.11c LDL-C(mmol/L) 0.52±0.02a 0.60±0.02b 0.68±0.04c 0.51±0.02a 0.46±0.05a HDL-C/LDL-C 6.02±1.13ab 5.02±0.56ab 3.81±0.70a 6.45±1.40b 7.11±1.81b

503 504 505 506


28


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Page 29 of 36


507 508

Table 6 Liver fatty acids (FA) compositions of triacylglycerols (TG), phospholipids (PL), and total lipids in mice fed five different diets at week six. Lipids (mg/mice ) Control 1 Control 2 High Fat CCSO MLCT Total 10:0 ND ND ND 5.69±1.04b 1.99±0.71a 12:0 ND ND ND 2.43±0.72b 0.98±0.02a 16:0 17.0±2.16a 116±5.25d 126±12.2d 82.3±7.05c 64.6±6.03b a c d b 18:0 8.45±1.08 69.4±0.11 105±9.03 46.2±3.09 48.0±6.41b 14.4±4.27a 116±8.25bc 130±16.2c 96.3±6.07b 110±10.1bc 18:1n-9 18:2n-6 20.7±5.47a 128±11.35d 131±13.3d 103±9.01c 59.1±3.35b a b b b 18:3n-3 0.53±0.02 5.09±1.01 6.40±1.12 4.70±0.18 5.35±1.44b 20:4n-6 9.98±1.09a 69.2±9.07b 102±8.02c 58.4±2.07b 62.4±8.23b 22:6n-3 9.40±1.31a 45.6±5.05d 38.6±5.01d 22.7±3.01b 29.2±2.03c a c d b Sum 80.4±5.16 549±47.1 640±55.3 422±31.3 382±34.2b TG 10:0 ND ND ND 3.71±0.06b 0.92±0.01a b 12:0 ND ND ND 1.18±0.03 0.31±0.28a 16:0 5.24±0.32a 59.6±4.04cd 68.7±7.47d 47.4±8.16c 33.1±9.36b 18:0 0.55±0.28a 11.3±5.05b 27.5±3.02c 19.9±2.85b 16.2±5.52b a c d b 5.52±1.79 62.7±8.04 88.2±6.09 42.0±6.01 57.3±8.01c 18:1n-9 9c12c18: 2n-6 7.72±2.07a 70.7±8.08d 89.8±8.31e 42.5±2.03c 32.5±6.02b a c d b 18:3n-3 0.24±0.03 3.11±0.01 3.20±0.01 2.26±0.04 2.25±1.02b 20:4n-6 0.95±0.01a 16.9±3.09b 26.8±0.51c 21.6±5.03b 18.5±0.04b 22:6n-3 1.64±0.06a 14.4±4.03bc 21.4±6.25c 16.7±0.01bc 13.1±0.07b a c d b Sum 21.9±6.37 239±19.1 326±20.1 197±9.07 174±8.43b PL 10:0 ND ND ND 0.16±0.01a 0.29±0.13b 12:0 ND ND ND 0.09±0.02 0.06±0.03 16:0 7.68±2.06a 41.4±0.01c 46.4±10.3c 19.5±3.36b 21.0±7.41b 18:0 7.38±0.23a 46.6±4.66c 68.7±6.43d 24.0±4.58b 30.6±8.10b a c c b 18:1n-9 3.51±1.18 20.5±0.36 25.4±8.18 7.92±1.28 18.3±5.35c 9c12c18: 2n-6 3.55±0.08a 18.1±2.02d 24.3±5.61d 7.93±0.31b 11.0±2.44c a c c b 18:3n-3 0.16±0.18 0.98±0.12 1.27±0.27 0.61±0.07 1.41±0.23c 20:4n-6 8.00±2.71a 46.8±7.01c 67.5±8.09d 24.0±7.03b 36.5±4.03c 22:6n-3 2.23±0.04a 10.0±3.30d 5.23±0.03b 4.61±0.71b 5.78±0.25c a d e b Sum 32.5±5.72 184±11.3 239±17.1 88.9±6.02 125±6.07c

509 510 511 512


29



513 514

Table 7 Fecal fatty acids (FA) compositions of triacylglycerols (TG), phospholipids (PL), and total lipids in mice fed five different diets at 6 weeks. FA (mg/mice/day) Control 1 Control 2 High Fat CCSO MLCT Total 10:0 ND ND ND 3.15±0.71 2.00±0.13 b 12:0 ND ND ND 1.78±0.02 1.00±0.03a 16:0 1.54±0.36d 0.76±0.12c 0.38±0.03a 0.50±0.06b 0.52±0.05b 18:0 0.38±0.03c 0.28±0.01b 0.16±0.05a 0.18±0.07ab 0.22±0.06ab b 2.21±0.35 1.36±0.02a 0.88±0.11a 1.2±0.30a 3.72±0.04c 18:1n-9 18:2n-6 3.12±0.01d 1.42±0.22c 0.50±0.02a 1.12±0.02b 0.93±0.22b 18:3n-3 0.19±0.05c 0.12±0.04b 0.04±0.02a 0.12±0.03b 0.07±0.01ab 20:4n-6 0.05±0.04 0.02±0.01 0.02±0.00 0.03±0.01 0.02±0.01 22:6n-3 0.17±0.02b 0.04±0.02a 0.02±0.01a 0.03±0.01a 0.03±0.00a Sum 7.66±1.06b 4.00±0.03a 2.00±0.01a 8.11±1.36b 8.51±1.44b TG 10:0 ND ND ND 0.14±0.03b 0.07±0.01a 12:0 ND ND ND 0.19±0.05b 0.07±0.01a c a a 16:0 0.06±0.00 0.02±0.01 0.02±0.01 0.02±0.01a 0.04±0.00b 18:0 0.01±0.01 0.01±0.00 0.01±0.01 0.01±0.01 0.01±0.01 a a a a 0.12±0.02 0.06±0.03 0.07±0.07 0.08±0.03 0.34±0.03b 18:1n-9 b a a a 18:2n-6 0.24±0.10 0.07±0.07 0.03±0.01 0.06±0.02 0.08±0.06a 18:3n-3 0.01±0.00b 0.01±0.00b 0.00±0.00a 0.01±0.00b 0.01±0.01b 20:4n-6 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 22:6n-3 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 0.00±0.00 Sum 0.44±0.12ab 0.17±0.08a 0.13±0.07a 0.51±0.25b 0.62±0.18b PL 10:0 ND ND ND 0.02±0.01 0.02±0.01 12:0 ND ND ND 0.03±0.01 0.01±0.02 16:0 0.11±0.06 0.09±0.05 0.07±0.03 0.08±0.05 0.07±0.03 18:0 0.03±0.01 0.04±0.02 0.04±0.03 0.06±0.06 0.04±0.00 0.09±0.01a 0.07±0.06a 0.10±0.41b 0.10±0.02ab 0.18±0.04b 18:1n-9 18:2n-6 0.09±0.05 0.07±0.01 0.04±0.01 0.08±0.03 0.06±0.01 18:3n-3 0.01±0.01 0.01±0.01 0.00±0.00 0.01±0.00 0.00±0.00 20:4n-6 0.02±0.00 0.01±0.01 0.01±0.00 0.01±0.01 0.01±0.01 b ab ab ab 22:6n-3 0.02±0.01 0.01±0.00 0.01±0.00 0.01±0.01 0.00±0.00a a a a b Sum 0.37±0.13 0.30±0.05 0.27±0.03 0.39±0.08 0.39±0.10b

515 516 517 518

Data are expressed as the mean ± SD. Mean values in a row with different letters (a, b, c, d) differ significantly, p < 0.05.

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Table 8 Effects of five different diets on liver biochemical variables of C57BL/6J mice during the entire experiments. Control 1 Control 2 High Fat CCSO MLCT a b c b 43.2±3.2 46.5±2.0 50±4.3 45.7±5.5 50.6± 4.3c Protein (μg/g liver) TG (mmol/mg protein) 0.39±0.01ab 0.43±0.02b 0.63±0.10c 0.32±0.06a 0.35±0.03ab a b c a 16.8±1.4 19.9±1.7 31.5±2.6 14.6±0.8 17.7±3.1ab TG (μmol/g liver) TC (mmol/mg protein) 0.2±0.01b 0.25±0.08c 0.32±0.02d 0.17±0.33a 0.20±0.03ab 8.64±0.57a 11.6±0.31b 16±2.36c 7.76±0.63a 10.1±1.03b TC (μmol/g liver) ApoA1(ng/mg protein) 15.5±2.02bc 13.4±1.24ab 12.9±0.95a 17.4±0.42cd 18.2±1.36d Ap7oB ng/mg protein) 27.9±0.88b 30.1±0.16c 40.2±0.37d 27.5±0.32b 26.3±0.56a ApoA1/ApoB 0.55±0.15bc 0.45±0.01ab 0.32±0.10a 0.63±0.03bc 0.69±0.08c

519 520

521 522 523


524

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525 526

Table 9 The levels of enzymes related to lipid metabolism in adipose tissue of C57BL/6J mice fed five different diets.

cAMP (pg/mg) PKA (ng/mg ) HSL (ng/mg) ATGL (ng/mg ) 527 528 529

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Control 1

Control 2

High Fat

CCSO

MLCT

1176±101bc

1027±100ab

843±138a

1361±129c

1352±145c

19.1±1.30c

16.5±0.22b

13.1±0.14a

20.9±0.76d

20.0±0.40cd

1.03±0.14ab

0.91±0.00a

0.84±0.11a

1.20±0.09b

1.18±0.13b

0.60±0.03bc

0.51±0.02a

0.47±0.05a

0.65±0.07c

0.62±0.09bc


530

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531

Figure Captions

532

Figure 1 TAG of CCSO, CO and interesterified product. (peak top number represents ECN of the

533

triaclyglycerol group).

534

Figure 2 Effect of different diets on HE staining of differentiated adipose tissues. Photomicgraphs

535

of HE-stained tissues(100×magnification) showing adipocytes fed (A) control 1 diet; (B) control 2

536

diet; (C) high fat diet; (D) CCSO diet; (E) MLCT diet for six weeks.

537

Figure 3 TOC Graph Investigation of lipid metabolism by a new structured lipid with medium

538

and long-chain triacylglycerols from Cinnamomum camphora seed oil in healthy C57BL/6J mice

539 540

33



Figure 1 TAG of CCSO, CO and interesterified product. (peak top number represents ECN of the triaclyglycerol group).

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A) Control 1

B) Control 2

C) High fat group

D) CCSO group

E ) MLCT group

Figure 2 Effect of different diets on HE staining of differentiated adipose tissues. Photomicgraphs of HE-stained tissues(100×magnification) showing adipocytes fed (A) control 1 diet; (B) control 2 diet; (C) high fat diet; (D) CCSO diet; (E) MLCT diet for six weeks.

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Figure 3 TOC Graph Investigation of lipid metabolism by a new structured lipid with medium and long-chain triacylglycerols from Cinnamomum camphora seed oil in healthy C57BL/6J mice

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Investigation of lipid metabolism by a new structured lipid with medium

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